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α-L -fucosidase in the reproductive organs and seminal plasma of the bull
Biochimica etBiophysicaActa 880 (1986) 91-95
91
Elsevier BBA 20110
BBA Report
a-L-Fucosidase in the reproductive organs and seminal plasma of the bull
Anneli Jauhiainen and Tapani Vanha-Perttula Department of Anatomy, Universityof Kuopio, P.O. Box 6, SF-70211 Kuopio (Finland) (Received July 19th, 1985)
Key words: a-L-Fucosidase; lsozyme separation; (Bull semen)
a-L-Fucosidase (EC 3.2.1.51) activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of a-L-fucosidase was found in the epididymis. Gel filtration at pH 7.0 revealed two a-L-fucosidases (a-L-fucosidase I and a-L-fucosidase I!) in most reproductive tissues, but seminal plasma, spermatozoa and epididymal cauda contained only form I. Fractionation at basic pH (pH 8.5) resulted in the elution of a-L-fucosidase as form II. Some differences were encountered in pH profiles and thermal stabilities of the two enzyme forms and they showed additional polymorphism after chromatofocusing. The comparison of enzyme profiles after fractionations suggests that cauda epididymidis is the main source of the seminal plasma activity in the bull.
The lysosomal hydrolase a-L-fucosidase ( a - L fucoside fucohydrolase, EC 3.2.1.51) is known to belong to a widely distributed group of glycosidases and to catalyse the hydrolysis of ct-Lfucose-containing biological substances, e.g. glycoproteins, glycolipids and oligosaccharides [1-3]. Several authors have found two forms of a-Lfucosidase separable by gel filtration or ion-exchange chromatography [1,4,5]. From these, ot-Lfucosidase I is a macromolecular form, which is adsorbed on DEAE-cellulose; a-L-fucosidase II has a lower molecular weight and it is not adsorbed on ion-exchange columns. Form II has also been found more sensitive to thermal inactivation than form I [1,4,5]. Treatment with neuraminidase leads to a decreased number of acidic isoenzymes and increases the more neutral forms, thus supporting the assumption of sialic acid as a structural component [6-11]. In the present paper we describe the separation of Ct-L-fucosidase isoenzymes from seminal plasma, spermatozoa and different reproductive organs of the bull using gel filtration on Sepharose 6B and chromatofocusing on a pH gradient.
The origin of bovine tissue and seminal plasma samples, their preparation for gel filtration and chromatofocusing and details of enzyme assays have been reported previously [12,13]. a-LFucosidase was measured spectrophotometrically with p-nitrophenyl-a-L-fucopyranoside (Sigma Chemical Co., St. Louis, MO, U.S.A.) as substrate and the activity is given as absorbance at 410 nm (fractionations) or as hydrolysis rates ( n m o l / m i n per mg protein) in tissue homogenates. a-L-Fucosidase activity in bull reproductive organs, seminal plasma and spermatozoa is shown in Table I and the hydrolysis rate of this enzyme in different epididymal segments (E~-E6) of immature (about 2 months of age), young (5-7 months of age) and adult (about 1 year of age) bulls as well as in epididymal cells, secretions and spermatozoa of the adult bulls is given in Table II. In the adult animals the highest level of a-Lfucosidase was found in the epididymis and particularly in caput segments E 2 + E 3 and in distal cauda segment E 6. In the epididymis, et-L-fUcosidase activity was mainly in cytosolic form (located in epididymal cells). However, a part of the activ-
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
92 TABLE I The specific activity ( n m o l / m i n per mg protein) of ~-Lfucosidase in the different reproductive organs, seminal plasma and ejaculated spermatozoa of adult (about 1 year of age) bulls ( n = 5). Values are given as means _+S.D. of duplicate measurements. Sample
a-L-Fucosidase activity ( n m o l / m i n per mg protein)
Testis Caput epididymidis Corpus epididymidis Cauda epididymidis Vas deferens Ampulla Seminal vesicle Seminal vesicle secretion Prostate Cowper's gland Seminal plasma Spermatozoa
2.91 _+0.38 14.94 _+0.82 9.78 _+0.42 18.02 _+0.98 1.99 + 0.49 1.03 -+ 0.11 1.50 -+ 0.28 0.08 -+ 0.01 1.77 -+ 0.62 3.32 _+0.44 3.87 _+2.02 12.69 _+1.78
ity seemed to be in the secretion of the cauda epididymidis. Also the epididymal and ejaculated spermatozoa contained high levels of c~-L-fucosidase. In immature animals the enzyme activity in
epididymis was significantly lower than in adult, mature animals. Further, the most dramatic rise in enzyme activity between young and adult bulls was seen in segments E 2 + E 3 and E 6. Gel filtration at pH 7.0 of bull testis, the caput part of the epididymis and also seminal vesicle and its secretion, prostate and Cowper's gland resulted in the appearance of two C~-L-fucosidase peaks GF-1 and GF-2 (Fig. 1). These activities generally eluted in fractions 114 128 ( M r = 3 3 0 0 0 0 ) and 134-150 ( M r = 200000), respectively. Only form GF-1 occurred in seminal plasma, spermatozoa and cauda epididymidis in gel filtration at pH 7.0. However, elution of the tissue homogenates or seminal plasma at more basic pH (pH 8.5) resulted in C~-L-fucosidase activity appearing at the position corresponding mostly to peak GF-2. Fractionation on chromatofocusing of bull seminal plasma and different reproductive organs resulted in the appearance of several activity peaks for c~-L-fucosidase. The p I values ranged from 7.2 to 5.2 and most of the activity was detected between p l 7.0 and 6.0. The pH optimum of peak GF-1 from gel filtra-
T A B L E II The specific activity ( n m o l / m i n per mg protein) of a-L-fucosidase in different segments of epididymis (E~ E6) in immature (about 2 months of age), young ( 5 - 7 months of age) and adult (about 1 year of age) bulls and in epididymal ceils, secretions and spermatozoa of different segments (E I, E 2 + E 3, E 6) in adult bulls. Values are reported as means_+ S.D. of duplicate measurements from the number of animals in parenthesis. Sample
EI
E2 E3 E4
E5 E6 Epididynal cells El E2 + E3
E6 Epididymal secretions E1 E2 + E3 E6
a-L-Fucosidase activity ( n m o l / m i n per mg protein) immature bulls
young bulls
1.81 +0.13 1.51 +0.22 1.63_+0.15 2.94_+ 1.10 5.49 _+0.06 6.32_+0.12
5.14_+ 1.13 5.16 _+3.17 11.45_+7.58 8.46 _+2.32 7.70 _+2.00 8.43_+0.68
(3) (3) (3) (3) (3) (3)
adult bulls (3) (3) (3) (3) (3) (3)
3.84_+ 1.07 12.33 _+3.37 16.29_+4.21 6.81 _+2.02 6.55 _+1.75 15.59_+2.58
(5) (5) (5) (5) (5) (5)
9.22 _+2.48 (3) 21.98 + 3.08 (3) 18.48_+6.38 (3) 1.05 _+0.32 (3) 2.10_+0.83 (3) 9.95 _+0.39 (3)
Epididymal spermatozoa El E2+ E3 E¢,
15.77_+3.37 (3) 20.10 _+7.80 (3) 20.66 + 5.21 (3)
93
tion was about 5.25 and that of peak GF-2 about 5.25-5.5. GF-1 also showed higher activity at more acidic pH values relative to GF-2. The enzyme GF-2 was more sensitive to thermal inactivationat 40-70°C than form GF-1 (Fig. 2). Both enzyme forms were equally sensitive to various divalent cations (Cd 2+, Cu 2+, Hg 2+, Mg 2+, Ni 2+, Pb 2+, Zn2+), while thiol compounds (cysteine, dithioerythritol) and chelating agents (EDTA, o-phenanthroline) did not cause any significant change at 1 mmol/1 concentration. The high level of a-L-fucosidase in the bovine epididymis supports the concept that this enzyme is linked with the functions of this organ in the fertile animal. The epididymal enzyme activity seemed to be partly in secretory ( E 6 ) and partly in non-secretory bound form (El-E6). Bostr~m and Ockerman [14] have found that a-L-fucosidase
levels are somewhat higher in the human epididymis than in other reproductive organs. Gill and Sutton [10], in contrast, have recently suggested that a-L-fucosidase activity in human seminal plasma may be produced by some of the accessory reproductive glands. The epididymal and ejaculated spermatozoa of the bull contained high levels of a-L-fucosidase. It has also been reported previously that goat, buffalo, bull and human sperm extracts contain significant amounts of a-L-fucosidase and in goat the enzyme concentration is even higher in sperm extracts than in seminal plasma [15]. Srivastava and collaborators [16] studied hydrolytic enzymes of chimpanzee semen and found that a larger amount of a-L-fucosidase activity can be extracted from spermatozoa with detergents (Hyamine and Triton) than with the MgC12 treatment. Zahler and
I
1.5
GEL FILTRATION eEl pH?.O o El SECRETION pH 7.0
• TESTIS pH 70 o TESTIS 131485
1.0
~0.5
< 1.0
• E6 pH70 o ~ pH85
• SEMINAL PLASMA pH 7.0 o SPERMATOZOA pH 7.0
0.5
110
130
150 0 90 FRACTION NUMBER
110
130
150
Fig. 1. a-L-Fucosidase activity of bull testis (pH 7.0, pH 8.5), E t and E 1 secretion (pH 7.0), E 6 (pH 7.0, pH 8.5), seminal plasma and spermatozoa (pH 7.0) after gel filtration on Sepharose 6B. The hydrolysis was measured in the fractions and the activities are given as absorbances at 410 nm.
94
O GF-I • GF-2 100 >>
~- 60 Z iii
20
tion-disaggregation is pH-dependent [22,23]. In all cases the polymeric forms have been more resistant to thermal treatment and low pH values [1,4,5,20], but additionally the physical state of the enzyme may influence its affinity (K,1) and specificity for substrates [5,22,24]. Increased sialylation has also been regarded as promoting aggregation [21]. In the bull cauda epididymidis, seminal plasma and spermatozoa the larger c~-Lfucosidase appears to be functionally more feasible than the smaller form. Otherwise the relationships of the two forms have not yet been fully understood. This work was supported by the Ministry of Agriculture and Forestry of Finland and the Federation of the Finnish AI Societies. References
3740 50 60 70 TEMPERATURE (°C) Fig. 2. Effect of temperature on the pooled c~-l.-fucosidase activities after gel filtration of bull testis (GF-1 and GF-2). The incubations were carried out in 0.1 M sodium acetate buffer. pH 5.25. The results are given as percentages of the uninhibited control values for the appropriate activities.
Doak [17] isolated the outer acrosomal membranes from bull spermatozoa by homogenization and subsequent discontinuous sucrose gradient. They found that the soluble fraction contained the highest c~-z,-fucosidase activity. This indicates that the enzyme is loosely attached to the membranes. Part of the sperm activity may actually be due to epididymal secretory enzyme bound to the plasma membrane. Gel filtration at pH 7.0 showed that seminal plasma, spermatozoa and the epididymal cauda had only the high-molecular-weight form of a-Lfucosidase. Thus, it is obvious that the seminal plasma enzyme originates mainly from cauda epididymidis. However, at a more basic pH (pH 8.5) this form of a-L-fucosidase could be dissociated into a smaller form, which had a slightly different pH curve profile and was more sensitive to thermal treatment. Previous studies with other tissues have indicated that the enzyme appears both as polymeric ( M r 145 000-390 000) and monomeric ( M r 35000-74000) forms [4,18-22] and the aggrega-
1 Wiederschain, G.Y., Kolibaba, L.G. and Rosenfeld, E.L. (1973) Clin. Chim. Acta 46, 305-310 2 Alhadeff, J.A. (1981) in Lysosomes and Lysosomal Storage Diseases (Callahan, J.W. and Lowden, J.A., eds.), pp. 299-314, Raven Press, New York 3 Beyer, E.M. and Wiederschain, G.Y. (1982) Clin. Chim. Acta 123. 251-259 4 Robinson, D. and Thorpe, R. (1973) Clin. Chim. Acta 47, 403-407 5 Di Matteo, G., Orfeo, M.A. and Romeo, G. (1976) Biochim. Biophys. Acta 429, 527-537 6 Turner, B.M., Beratis, N.G., Turner, V.S. and Hirschhorn, K. (1974) Clin. Chim. Acta 57, 29-35 7 Alhadeff, J.A., Tennant, L. and O'Brien, J.S. (1975) Dev. Biol. 47, 319 324 8 Di Matteo, G., Durand, P., Gatti, R., Maresca. A., Orfeo, M., Urbano, F. and Romeo, G. (1976) Biochim. Biophys. Acta 429, 538-545 9 Alhadeff, J.A., Cimino, G. and Janowsky, A. (1978) Mol. Cell. Biochem. 19, 171-180 10 Gill, P. and Sutton, J.G. (1984) Hum. Hered. 34, 231-239 11 Ho, M.W., Norden, A.G.W., Alhadeff, J.A. and O'Brien, J.S. (1977) Mol. Cell. Biochem. 17, 125-140 12 Jauhiainen, A. and Vanha-Perttula, T. (1985) J. Reprod. Fertil. 74, 669-680 13 Jauhiainen, A. and Vanha-Perttula, T. (1986) J. Reprod. Fertil. 76, 000-000 14 Bostr6m, K. and Ockerman, P.-A. (1971) Scand. J. Urol. Nephrol. 5, 117-122 15 Khar, A. and Anand, S.R. (1974) Indian J. Biochem. Biophys. 11, 88 89 16 Srivastava, P.N., Farooqui, A.A. and Gould, K,G. (1981) Biol. Reprod. 25, 363-369 17 Zahler, W.L. and Doak, G.A. (1975) Biochim Biophys. Acta 406, 479-488 18 Carlsen, R.B. and Pierce, J.G. (1972) J. Biol. Chem. 247, 23-32
95 19 Opheim, D.J. and Touster, O. (1977) J. Biol. Chem. 252, 739-743 20 Alam, T. and Balasubramanian, A.S. (1978) Biochim. Biophys. Acta 524, 373-384 21 Alhadeff, J.A. and Janowsky, A.J. (1978) Clin. Chim. Acta 82, 133-140
22 DiCioccio, R.A., Barlow, J.J. and Matta, K.L. (1982) J. Biol. Chem. 257, 714-718 23 Willems, P.J., Romeo, E., den Tandt, W.R., Van Elsen, A.F. and Leroy, J.G. (1981) Hum. Genet. 59, 115-118 24 Bernard, M., Percheron, F. and Foglietti, M.J. (1983) Int. J. Biochem. 15, 1385-1387
91
Elsevier BBA 20110
BBA Report
a-L-Fucosidase in the reproductive organs and seminal plasma of the bull
Anneli Jauhiainen and Tapani Vanha-Perttula Department of Anatomy, Universityof Kuopio, P.O. Box 6, SF-70211 Kuopio (Finland) (Received July 19th, 1985)
Key words: a-L-Fucosidase; lsozyme separation; (Bull semen)
a-L-Fucosidase (EC 3.2.1.51) activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of a-L-fucosidase was found in the epididymis. Gel filtration at pH 7.0 revealed two a-L-fucosidases (a-L-fucosidase I and a-L-fucosidase I!) in most reproductive tissues, but seminal plasma, spermatozoa and epididymal cauda contained only form I. Fractionation at basic pH (pH 8.5) resulted in the elution of a-L-fucosidase as form II. Some differences were encountered in pH profiles and thermal stabilities of the two enzyme forms and they showed additional polymorphism after chromatofocusing. The comparison of enzyme profiles after fractionations suggests that cauda epididymidis is the main source of the seminal plasma activity in the bull.
The lysosomal hydrolase a-L-fucosidase ( a - L fucoside fucohydrolase, EC 3.2.1.51) is known to belong to a widely distributed group of glycosidases and to catalyse the hydrolysis of ct-Lfucose-containing biological substances, e.g. glycoproteins, glycolipids and oligosaccharides [1-3]. Several authors have found two forms of a-Lfucosidase separable by gel filtration or ion-exchange chromatography [1,4,5]. From these, ot-Lfucosidase I is a macromolecular form, which is adsorbed on DEAE-cellulose; a-L-fucosidase II has a lower molecular weight and it is not adsorbed on ion-exchange columns. Form II has also been found more sensitive to thermal inactivation than form I [1,4,5]. Treatment with neuraminidase leads to a decreased number of acidic isoenzymes and increases the more neutral forms, thus supporting the assumption of sialic acid as a structural component [6-11]. In the present paper we describe the separation of Ct-L-fucosidase isoenzymes from seminal plasma, spermatozoa and different reproductive organs of the bull using gel filtration on Sepharose 6B and chromatofocusing on a pH gradient.
The origin of bovine tissue and seminal plasma samples, their preparation for gel filtration and chromatofocusing and details of enzyme assays have been reported previously [12,13]. a-LFucosidase was measured spectrophotometrically with p-nitrophenyl-a-L-fucopyranoside (Sigma Chemical Co., St. Louis, MO, U.S.A.) as substrate and the activity is given as absorbance at 410 nm (fractionations) or as hydrolysis rates ( n m o l / m i n per mg protein) in tissue homogenates. a-L-Fucosidase activity in bull reproductive organs, seminal plasma and spermatozoa is shown in Table I and the hydrolysis rate of this enzyme in different epididymal segments (E~-E6) of immature (about 2 months of age), young (5-7 months of age) and adult (about 1 year of age) bulls as well as in epididymal cells, secretions and spermatozoa of the adult bulls is given in Table II. In the adult animals the highest level of a-Lfucosidase was found in the epididymis and particularly in caput segments E 2 + E 3 and in distal cauda segment E 6. In the epididymis, et-L-fUcosidase activity was mainly in cytosolic form (located in epididymal cells). However, a part of the activ-
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
92 TABLE I The specific activity ( n m o l / m i n per mg protein) of ~-Lfucosidase in the different reproductive organs, seminal plasma and ejaculated spermatozoa of adult (about 1 year of age) bulls ( n = 5). Values are given as means _+S.D. of duplicate measurements. Sample
a-L-Fucosidase activity ( n m o l / m i n per mg protein)
Testis Caput epididymidis Corpus epididymidis Cauda epididymidis Vas deferens Ampulla Seminal vesicle Seminal vesicle secretion Prostate Cowper's gland Seminal plasma Spermatozoa
2.91 _+0.38 14.94 _+0.82 9.78 _+0.42 18.02 _+0.98 1.99 + 0.49 1.03 -+ 0.11 1.50 -+ 0.28 0.08 -+ 0.01 1.77 -+ 0.62 3.32 _+0.44 3.87 _+2.02 12.69 _+1.78
ity seemed to be in the secretion of the cauda epididymidis. Also the epididymal and ejaculated spermatozoa contained high levels of c~-L-fucosidase. In immature animals the enzyme activity in
epididymis was significantly lower than in adult, mature animals. Further, the most dramatic rise in enzyme activity between young and adult bulls was seen in segments E 2 + E 3 and E 6. Gel filtration at pH 7.0 of bull testis, the caput part of the epididymis and also seminal vesicle and its secretion, prostate and Cowper's gland resulted in the appearance of two C~-L-fucosidase peaks GF-1 and GF-2 (Fig. 1). These activities generally eluted in fractions 114 128 ( M r = 3 3 0 0 0 0 ) and 134-150 ( M r = 200000), respectively. Only form GF-1 occurred in seminal plasma, spermatozoa and cauda epididymidis in gel filtration at pH 7.0. However, elution of the tissue homogenates or seminal plasma at more basic pH (pH 8.5) resulted in C~-L-fucosidase activity appearing at the position corresponding mostly to peak GF-2. Fractionation on chromatofocusing of bull seminal plasma and different reproductive organs resulted in the appearance of several activity peaks for c~-L-fucosidase. The p I values ranged from 7.2 to 5.2 and most of the activity was detected between p l 7.0 and 6.0. The pH optimum of peak GF-1 from gel filtra-
T A B L E II The specific activity ( n m o l / m i n per mg protein) of a-L-fucosidase in different segments of epididymis (E~ E6) in immature (about 2 months of age), young ( 5 - 7 months of age) and adult (about 1 year of age) bulls and in epididymal ceils, secretions and spermatozoa of different segments (E I, E 2 + E 3, E 6) in adult bulls. Values are reported as means_+ S.D. of duplicate measurements from the number of animals in parenthesis. Sample
EI
E2 E3 E4
E5 E6 Epididynal cells El E2 + E3
E6 Epididymal secretions E1 E2 + E3 E6
a-L-Fucosidase activity ( n m o l / m i n per mg protein) immature bulls
young bulls
1.81 +0.13 1.51 +0.22 1.63_+0.15 2.94_+ 1.10 5.49 _+0.06 6.32_+0.12
5.14_+ 1.13 5.16 _+3.17 11.45_+7.58 8.46 _+2.32 7.70 _+2.00 8.43_+0.68
(3) (3) (3) (3) (3) (3)
adult bulls (3) (3) (3) (3) (3) (3)
3.84_+ 1.07 12.33 _+3.37 16.29_+4.21 6.81 _+2.02 6.55 _+1.75 15.59_+2.58
(5) (5) (5) (5) (5) (5)
9.22 _+2.48 (3) 21.98 + 3.08 (3) 18.48_+6.38 (3) 1.05 _+0.32 (3) 2.10_+0.83 (3) 9.95 _+0.39 (3)
Epididymal spermatozoa El E2+ E3 E¢,
15.77_+3.37 (3) 20.10 _+7.80 (3) 20.66 + 5.21 (3)
93
tion was about 5.25 and that of peak GF-2 about 5.25-5.5. GF-1 also showed higher activity at more acidic pH values relative to GF-2. The enzyme GF-2 was more sensitive to thermal inactivationat 40-70°C than form GF-1 (Fig. 2). Both enzyme forms were equally sensitive to various divalent cations (Cd 2+, Cu 2+, Hg 2+, Mg 2+, Ni 2+, Pb 2+, Zn2+), while thiol compounds (cysteine, dithioerythritol) and chelating agents (EDTA, o-phenanthroline) did not cause any significant change at 1 mmol/1 concentration. The high level of a-L-fucosidase in the bovine epididymis supports the concept that this enzyme is linked with the functions of this organ in the fertile animal. The epididymal enzyme activity seemed to be partly in secretory ( E 6 ) and partly in non-secretory bound form (El-E6). Bostr~m and Ockerman [14] have found that a-L-fucosidase
levels are somewhat higher in the human epididymis than in other reproductive organs. Gill and Sutton [10], in contrast, have recently suggested that a-L-fucosidase activity in human seminal plasma may be produced by some of the accessory reproductive glands. The epididymal and ejaculated spermatozoa of the bull contained high levels of a-L-fucosidase. It has also been reported previously that goat, buffalo, bull and human sperm extracts contain significant amounts of a-L-fucosidase and in goat the enzyme concentration is even higher in sperm extracts than in seminal plasma [15]. Srivastava and collaborators [16] studied hydrolytic enzymes of chimpanzee semen and found that a larger amount of a-L-fucosidase activity can be extracted from spermatozoa with detergents (Hyamine and Triton) than with the MgC12 treatment. Zahler and
I
1.5
GEL FILTRATION eEl pH?.O o El SECRETION pH 7.0
• TESTIS pH 70 o TESTIS 131485
1.0
~0.5
< 1.0
• E6 pH70 o ~ pH85
• SEMINAL PLASMA pH 7.0 o SPERMATOZOA pH 7.0
0.5
110
130
150 0 90 FRACTION NUMBER
110
130
150
Fig. 1. a-L-Fucosidase activity of bull testis (pH 7.0, pH 8.5), E t and E 1 secretion (pH 7.0), E 6 (pH 7.0, pH 8.5), seminal plasma and spermatozoa (pH 7.0) after gel filtration on Sepharose 6B. The hydrolysis was measured in the fractions and the activities are given as absorbances at 410 nm.
94
O GF-I • GF-2 100 >>
~- 60 Z iii
20
tion-disaggregation is pH-dependent [22,23]. In all cases the polymeric forms have been more resistant to thermal treatment and low pH values [1,4,5,20], but additionally the physical state of the enzyme may influence its affinity (K,1) and specificity for substrates [5,22,24]. Increased sialylation has also been regarded as promoting aggregation [21]. In the bull cauda epididymidis, seminal plasma and spermatozoa the larger c~-Lfucosidase appears to be functionally more feasible than the smaller form. Otherwise the relationships of the two forms have not yet been fully understood. This work was supported by the Ministry of Agriculture and Forestry of Finland and the Federation of the Finnish AI Societies. References
3740 50 60 70 TEMPERATURE (°C) Fig. 2. Effect of temperature on the pooled c~-l.-fucosidase activities after gel filtration of bull testis (GF-1 and GF-2). The incubations were carried out in 0.1 M sodium acetate buffer. pH 5.25. The results are given as percentages of the uninhibited control values for the appropriate activities.
Doak [17] isolated the outer acrosomal membranes from bull spermatozoa by homogenization and subsequent discontinuous sucrose gradient. They found that the soluble fraction contained the highest c~-z,-fucosidase activity. This indicates that the enzyme is loosely attached to the membranes. Part of the sperm activity may actually be due to epididymal secretory enzyme bound to the plasma membrane. Gel filtration at pH 7.0 showed that seminal plasma, spermatozoa and the epididymal cauda had only the high-molecular-weight form of a-Lfucosidase. Thus, it is obvious that the seminal plasma enzyme originates mainly from cauda epididymidis. However, at a more basic pH (pH 8.5) this form of a-L-fucosidase could be dissociated into a smaller form, which had a slightly different pH curve profile and was more sensitive to thermal treatment. Previous studies with other tissues have indicated that the enzyme appears both as polymeric ( M r 145 000-390 000) and monomeric ( M r 35000-74000) forms [4,18-22] and the aggrega-
1 Wiederschain, G.Y., Kolibaba, L.G. and Rosenfeld, E.L. (1973) Clin. Chim. Acta 46, 305-310 2 Alhadeff, J.A. (1981) in Lysosomes and Lysosomal Storage Diseases (Callahan, J.W. and Lowden, J.A., eds.), pp. 299-314, Raven Press, New York 3 Beyer, E.M. and Wiederschain, G.Y. (1982) Clin. Chim. Acta 123. 251-259 4 Robinson, D. and Thorpe, R. (1973) Clin. Chim. Acta 47, 403-407 5 Di Matteo, G., Orfeo, M.A. and Romeo, G. (1976) Biochim. Biophys. Acta 429, 527-537 6 Turner, B.M., Beratis, N.G., Turner, V.S. and Hirschhorn, K. (1974) Clin. Chim. Acta 57, 29-35 7 Alhadeff, J.A., Tennant, L. and O'Brien, J.S. (1975) Dev. Biol. 47, 319 324 8 Di Matteo, G., Durand, P., Gatti, R., Maresca. A., Orfeo, M., Urbano, F. and Romeo, G. (1976) Biochim. Biophys. Acta 429, 538-545 9 Alhadeff, J.A., Cimino, G. and Janowsky, A. (1978) Mol. Cell. Biochem. 19, 171-180 10 Gill, P. and Sutton, J.G. (1984) Hum. Hered. 34, 231-239 11 Ho, M.W., Norden, A.G.W., Alhadeff, J.A. and O'Brien, J.S. (1977) Mol. Cell. Biochem. 17, 125-140 12 Jauhiainen, A. and Vanha-Perttula, T. (1985) J. Reprod. Fertil. 74, 669-680 13 Jauhiainen, A. and Vanha-Perttula, T. (1986) J. Reprod. Fertil. 76, 000-000 14 Bostr6m, K. and Ockerman, P.-A. (1971) Scand. J. Urol. Nephrol. 5, 117-122 15 Khar, A. and Anand, S.R. (1974) Indian J. Biochem. Biophys. 11, 88 89 16 Srivastava, P.N., Farooqui, A.A. and Gould, K,G. (1981) Biol. Reprod. 25, 363-369 17 Zahler, W.L. and Doak, G.A. (1975) Biochim Biophys. Acta 406, 479-488 18 Carlsen, R.B. and Pierce, J.G. (1972) J. Biol. Chem. 247, 23-32
95 19 Opheim, D.J. and Touster, O. (1977) J. Biol. Chem. 252, 739-743 20 Alam, T. and Balasubramanian, A.S. (1978) Biochim. Biophys. Acta 524, 373-384 21 Alhadeff, J.A. and Janowsky, A.J. (1978) Clin. Chim. Acta 82, 133-140
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